ORIGINAL CONTRIBUTION

Systemic Metabolic Abnormalities in Adult-onset

Acid Maltase Deficiency
Beyond Muscle Glycogen Accumulation
Juan M. Pascual, MD, PhD; Charles R. Roe, MD

Importance: The physiological relevance of acid malt-
ase (acid ␣-glucosidase, an enzyme that degrades lyso-
somal glycogen) is well recognized in liver and muscle.
In late (adult)–onset acid maltase deficiency (glycogen stor-
age disease type II [GSD II]), glycogen accumulates in-
side muscular lysosomes in the context of reduced enzy-
matic activity present not only in muscle, but also
throughout the organism. Yet, disease manifestations are
commonly attributed to lysosomal disruption and au-
tophagic vesicle buildup inside the myofiber due to a lack
of obvious hepatic or broader metabolic dysfunction. How-
ever, current therapies primarily focused on reducing gly-
cogen deposition by dietary or enzyme replacement have
not been consistently beneficial, providing the motiva-
tion for a better understanding of disease mechanisms.
Objective: To provide a systematic overview of me-
tabolism and methylation capacity using widely avail-
able analytical methods by evaluating secondary com-
promise of (1) the citric acid cycle, (2) methylation
capacity, and (3) nutrient sensor interaction in as many
as 33 patients with GSD II (ie, not all patients were avail-
able for all assessments) treated with only a low-
carbohydrate/high-protein, calorie-balanced diet.
Design, Setting, and Patients: Case series includ-
ing clinical and analytical characterization in an aca-
demic setting involving 33 enzymatically proved adults
with GSD II treated only with a low-carbohydrate/high-
protein, calorie-balanced diet.
Main Outcome and Measure: Biochemical analysis
of blood and urine samples.
Results: Patients exhibited evidence for disturbed en-
ergy metabolism contributing to a chronic catabolic state
and those who were studied further also displayed dimin-
ished plasma methylation capacity and elevated levels of
insulin-like growth factor type 1 and its carrier protein in-
sulin-like growth factor binding protein 3 (IGFBP-3).
Conclusions and Relevance: The simplest unifying
interpretation of these abnormalities is nutrient sensor
disturbance with secondary energy failure leading to a
chronic catabolic state. Data also provide the frame-
work for the investigation of potentially beneficial inter-
ventions, including methylation supplementation, as ad-
juncts specifically targeted to ameliorate the systemic
metabolic abnormalities of this disorder.
JAMA Neurol. 2013;70(6):756-763. Published online April
22, 2013. doi:10.1001/jamaneurol.2013.1507

Author Affiliations: Rare Brain
Disorders Clinic and Laboratory,
Departments of Neurology and
Neurotherapeutics (Drs Pascual
and Roe), Physiology and
Pediatrics (Dr Pascual), The
University of Texas Southwestern
Medical Center, Dallas.
G
LYCOGEN METABOLISM
occurs throughout the
organism, particularly in
organs that expend en-
ergy generating work and
maintaining metabolic homeostasis, such
as muscle and liver.1,2 Unlike other glyco-
genoses, late-onset glycogen storage dis-
ease type II (adult GSD II) is character-
ized by loss of function in all tissues of the
lysosomal enzyme, acid ␣-glucosidase
(acid maltase), which tends to exhibit
higher residual activity than in the infan-
tile form of the disorder.3 Despite this uni-
form deficiency, patients with GSD II do
not experience multiorgan failure. In-
stead, striated skeletal and sphincter
muscles (especially the diaphragm) are se-
verely affected, causing dysphagia, sphinc-
ter hypotonia, progressive limb weak-
ness, muscular atrophy, and ultimately,
respiratory failure.4,5
Elevations of serum enzymes (creatine
kinase [CK] and transaminases) and, oc-
casionally, decreases in plasma alanine and
glutamine levels, have generally been at-
tributed to the myopathy,6,7 such that there
has been no systematic focus on the po-
tential contribution of additional organ
derangement to the disorder.8 Conse-
quently, current management is centered
on limiting glycogen deposition in skel- Author Affi
etal muscle via dietary modification or en- Disorders C
zyme replacement (ERT). The current diet Department
Neurothera
includes reduced carbohydrate and in- and Roe), P
creased protein intake, often with alanine Pediatrics (D
supplementation and programmed physi- University o
cal exercise.6,7,9-12 This diet, combined with Medical Cen

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 exercise, reversed muscle glycogen accumulation in 2 af-
fected siblings11 and has retarded the rate of clinical de-
terioration.10 Following introduction of intravenous ERT,
clinical studies have not demonstrated homogeneous ben-
efit for patients with GSD II.13,14 The obvious corollary to
these observations is that therapy for GSD II may be in-
sufficient to provide the desired clinical benefit. From a
more critical perspective, they may also be taken to sug-
gest the existence of additional, unrecognized, and un-
corrected metabolic derangements (other than compro-
mised glycogen degradation) that may contribute to the
pathogenesis of this disorder and, perhaps, to an inad-
equate response to current therapies.13
In this study, we set out to provide a systematic over-
view of metabolism and methylation capacity using widely
available analytical methods by evaluating secondary com-
promise of (1) the citric acid cycle (CAC), (2) methyla-
tion capacity, and (3) nutrient sensor interaction in as
many as 33 patients (ie, not all patients were available
for all assessments) treated only with diet. We reasoned
that, if affected, these factors may reflect an underlying
energy-deficient state contributing to the progressive clini-
cal deterioration of these patients.
METHODS
Standard informed consent and ethical procedures were fol-
lowed. One patient received triheptanoin supplementation solely
for analytical purposes (rather than to test clinical efficacy) un-
der an instutional review board–approved protocol and Food and
Drug Administration IND 59303. Lysosomal ␣-glucosidase de-
ficiency was confirmed in all patients by leukocyte enzymatic
assay prompted by myopathic clinical features. All available pa-
tients known to us with disease onset after age 15 years were in-
cluded. They were sequentially enrolled during 1 year on the ba-
sis of clinical and enzymatic criteria. Not all patients were available
for participation in all analytical studies, and additional selec-
tion criteria were not imposed on those who were able to par-
ticipate in further study. Disease onset was defined as the time
when weakness or fatigability first led to general medical atten-
tion. Additional data included demographic profiles, ambula-
tory and ventilatory assistance, nutritional route, and Walton and
Gardner-Medwin14 and Slonim10 functional scores.
All blood assays were performed on plasma obtained from
(overnight) fasting samples. Methods for quantitative acylcar-
nitines, plasma amino acids, and urinary organic acids, and their
reference ranges have been described previously.15-17 Acylcar-
nitine profiles and plasma amino acid levels were obtained in
all 33 patients. Samples for urine organic acid analyses were
available in a subset of 19 patients. Blood chemistry analyses
included the following levels: glucose, serum urea nitrogen, cre-
atinine, aspartate aminotransferase, alanine aminotransferase,
and CK.
Total plasma homocysteine was measured by high-
performance liquid chromatography with fluorescence detec-
tion. 1 8 Plasma S-adenosylmethionine (SAM), and S-
adenosylhomocysteine (SAH) were measured in 3 patients by
a modification of the stable-isotope dilution liquid chromatog-
raphy–electrospray injection tandem mass spectrometry pre-
viously described.19 Plasma guanidinoacetic acid, and creatine
were measured in 7 patients by the Clinical Chemistry Depart-
ment of the Free University of Amsterdam, Amsterdam, the
Netherlands, using previously described methods.20
Plasma IGF-1, insulin-like growth factor binding pro-
tein-3 (IGFBP-3), and growth hormone (GH) were measured
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in all patients by D. S. Laboratories, Houston, Texas. The re-
sults were compared to age-related standards because both IGF-1
and IGFBP-3 decrease with age.
Statistical results were obtained with GraphPad Prism (ver-
sion 4.00; GraphPad Software, Inc) and tested using a non-
parametric Mann-Whitney test. Results were considered sta-
tistically significant at Pⱕ.05.
RESULTS
PATIENT CHARACTERISTICS
Patient age ranged from 15 to 72 years and included 15
females and 18 males whose symptom duration ranged
from 2 to 48 years (Table 1). They were sequentially
enrolled during 1 year on the basis of clinical and enzy-
matic criteria. As noted, not all patients were available
for participation in all analytical studies, and the tables
allow for correlative patient identification. Of the 33, at
the time of blood and urine testing, 10 required a wheel-
chair, 18 were ambulatory, and 5 were ambulatory but
required assistive devices. Ten patients (30%) required
respiratory support (8 nightly, 2 continuously). Frozen
samples for metabolic analysis (propionylcarnitine, ala-
nine, and glutamine) were obtained from all 33 pa-
tients. Urinary citrate was measured in 26 patients. All
patients received a standard low-carbohydrate/high-
protein, calorie-balanced diet.6 None of the patients had
received ERT or supplements, such as carnitine, ala-
nine, glutamine, or citrate. Although follow-up analy-
ses were beyond the scope of this study, 4 patients were
tested again after 7 months, to evaluate whether urinary
citrate excretion was subject to fluctuation.
ACYLCARNITINES AND RELATED ANALYTES
Quantitative results for blood propionylcarnitine, ala-
nine, glutamine, and urinary excretion of citrate are sum-
marized in Table 2 together with patient ages. No sig-
nificant or consistent abnormalities were noted in the
plasma amino acid or urinary organic acid analyses. Quan-
titative blood spot free carnitine and acylcarnitines (acetyl-
[C2]) to linoleoyl-carnitine (C18:2) were measured for all
patients. Propionylcarnitine was markedly reduced in most
patients. Using our isotope dilution method, levels less than
1.5 ␮M and, especially, less than 1.0 ␮M are rarely ob-
served in healthy participants in blood spot analyses
(C.R.R., unpublished data, August 18, 2012). Twenty-
three (70%) of 33 patients exhibited levels lower than 1.5
␮M (reference range, 0.28-1.42 ␮M) and 11 patients ex-
hibited levels lower than 1.0 ␮M (reference range, 0.28-
0.95 ␮M). All other acylcarnitine levels were consistently
normal (data not shown). Plasma amino acid analysis re-
vealed that only 3 patients exhibited reduced levels of ala-
nine or glutamine. Serum CK levels for all 33 patients
ranged from 58 to 1401 U/L (to convert units per liter to
microkatals per liter, multiply by 0.0167). Twenty-four pa-
tients (73%) exhibited elevated CK levels (⬎130 U/L).
URINARY METABOLITES
Urine samples for organic acid analysis were available from
26 patients. Only citrate levels demonstrated abnormali-
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 Table 1. Demographic and Clinical Characteristics of Patients With GSD II

Scores
Sex/Patient Disease Respiratory Nutrition CK Level, Walton and
Age, y Duration, y Mobility Support Route U/L Gardner-Medwin14 Slonim10
M/15 NA Ambulatory None Oral NA NA NA
F/27 9 Wheelchair None Oral 236 6 6
M/31 8 Ambulatory; uses cane None Oral 781 3 3
F/35 2 Ambulatory None Oral 917 6 6
M/35 Wheelchair Nocturnal Oral
M/37 8 Ambulatory None Oral 201 2 2
M/37 5 Ambulatory None Oral 604 2 2
F/39 16 Ambulatory; uses None Oral 111 4 4
rollator
F/40 9 Ambulatory Nocturnal Oral 238 3 3
F/41 5 Ambulatory None Oral 1113 3 3
M/41 11 Ambulatory None Oral 558 3 3
F/42 5 Wheelchair None Oral 969 4 4
M/42 NA Ambulatory None Oral NA NA NA
F/43 8 Ambulatory None Oral 968 1 1
M/45 10 Ambulatory None Oral 806 3 3
F/48 5 Wheelchair Nocturnal Oral 241 4 4
M/49 20 Ambulatory None Oral 150 1 1
F/51 8 Ambulatory None Oral 203 1 1
F/51 20 Wheelchair None Oral 1164 3 3
M/52 NA Ambulatory; uses cane NA NA NA NA NA
F/54 32 Wheelchair Nocturnal Oral 257 4 4
M/55 16 Ambulatory Nocturnal Oral 1401 3 3
M/56 25 Wheelchair Continuous Nasogastric 313 4 4
F/56 7 Ambulatory Nocturnal Oral 374 3 3
F/57 16 Ambulatory None Oral 494 3 3
M/58 NA NA NA NA NA NA NA
M/63 NA Wheelchair Nocturnal Oral 58 8 7
M/64 35 Ambulatory Nocturnal Oral 414 2 2
M/65 34 Ambulatory; uses None Oral 623 3 3
rollator
F/65 NA Bed bound Continuous NA NA NA NA
M/66 NA Ambulatory None NA NA NA NA
M/68 48 Wheelchair None Oral 195 4 4
F/72 10 Ambulatory None Oral 382 3 3

Abbreviations: CK, creatine kinase; GSD II, glycogen storage disease type II; and NA, not applicable.
SI conversion factor: To convert creatine kinase to microkatals per liter; multiply by 0.0167.

ties. Citrate levels from 12 patients (46%) were above nor-
mal. Eight of these were markedly increased (⬎1000
mmol/mol creatinine) (Figure 1A). Due to the wide dis-
tribution of citrate levels in these patients, the group, as
a whole, was not significantly different from healthy con-
trols (P = .09). However, comparison of the 8 patients with
citrate levels higher than 1000 mmol/mol creatinine re-
vealed a significant difference from healthy controls
(P ⱕ .001). Four of 8 patients were studied again 7 months
later. Initially, 3 of these 4 patients had urinary citrate
levels in the reference range (⬍803 mmol/mol creati-
nine). However, 7 months later, their urine citrate lev-
els had become elevated (Figure 1B), indicating that ci-
trate excretion was subject to considerable fluctuation.
No other organic acid abnormalities were noted to sug-
gest metabolic derangement in fat oxidation, amino acid
degradation, or vitamin deficiencies.
lytical testing (Figure 2). Plasma levels of IGF-1 and
IGFBP-3 gradually decrease with age. Therefore, pa-
tient results were compared with 3 healthy age groups
(30-40 years, 41-50 years, and 51-70 years). In compari-
son with the corresponding reference ranges for age, all
26 patients exhibited significant elevations in both IGF-1
and IGFBP-3. The IGF-1 levels were highly significant
for all age groups (P ⱕ .001). The comparison of IGFBP-3
levels with normal age ranges was also significant: P = .02
for the 30- to 40-year-old patients, and P ⱕ .001 for the
2 older age groups. Growth hormone levels were nor-
mal (⬍0.1 ng/mL) (to convert nanograms per milliliter
to nanomoles per liter, multiply by 0.131) for all 26 pa-
tients (data not shown).
RESPONSE TO STIMULATION
OF ANAPLEROSIS

IGF-1 AND IGFBP-3 LEVELS IN GSD II The previous data led to the hypothesis that, if IGF-1 and
IGFBP-3 levels were elevated as the result of energy de-
Plasma levels of IGF-1, IGFBP-3, and GH were mea- ficiency in GSD II, stimulation of anaplerosis (ie, replen-
sured in 26 patients who were available for further ana- ishment of CAC intermediates) might reduce both plasma

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 Table 2. Plasma and Urinary Metabolic Parameters in 33 Adult-onset Patients With GSD II

Patient Age, Propionylcarnitine, Alanine, Glutamine, Urinary Citrate, mmol/mol

y µM mg/dL mg/dL Creatinine
15 1.27 2.09 6.34 2287
27 0.60 2.25 8.11 NA
31 1.27 3.71 9.46 907
35 0.72 4.28 10.73 629
35 3.03 3.88 8.59 540
37 2.81 2.43 6.74 384
37 1.38 2.41 6.21 830
39 1.19 5.32 12.16 NA
40 1.34 4.20 13.07 799
41 1.08 3.75 12.50 14
41 0.92 4.07 10.54 NA
42 0.83 3.60 7.86 1490
42 1.25 1.15 6.28 847
43 0.45 1.74 4.87 1997
45 1.24 1.83 6.94 19
48 0.82 3.81 10.80 NA
49 1.56 7.00 12.41 13
51 2.91 4.06 10.44 NA
51 0.90 4.97 10.54 1251
52 1.36 1.44 6.20 314
54 0.28 1.41 7.15 69
55 1.72 3.28 5.01 NA
56 0.67 2.89 8.08 1262
56 2.50 2.58 6.81 1196
57 1.10 2.86 7.10 1292
58 2.54 2.30 6.45 213
63 1.68 4.57 9.19 163
64 0.95 2.81 7.47 1257
65 3.19 5.18 9.02 163
65 1.42 3.19 9.51 1220
66 1.32 2.92 9.67 878
68 2.54 3.16 8.10 NA
72 0.90 5.78 8.30 17
Reference levels ⬍2.71 1.44-5.10 6.20-10.52 186-803

Abbreviations: GSD II, glycogen storage disease type II; NA, not applicable.
SI conversion factor: To convert alanine to micromoles per liter; multiply by 112.2; and glutamine to micromoles per liter, multiply by 68.423.

IGF-1 and IGFBP-3 levels and reduce urinary citrate ex-

cretion. To test this hypothesis, the effects of a single oral A B
triheptanoin meal (an anaplerotic triglyceride21-23) were 3500 3500
Urine Citrate Level, mmol/mol Creatinine

tested in 1 patient. This 65-year-old man was given a single 3000 3000
dose of triheptanoin (0.25 g/kg) in 80 g of low-fat/low-
carbohydrate yogurt. Plasma IGF-1, IGFBP-3, GH, and 2500 2500
P = .09
urinary citrate were measured over 6 hours following the 2000 2000
meal. The IGF-1 levels decreased by 23%, from 213 ng/mL 1500 1500
to a normal mean level of 163 ng/mL (IGF-1 reference,
1000 1000
154[ 49] ng/mL) (Figure 3A). The IGFBP-3 levels also
decreased to a normal level for age: from 3672 to 3345 500 500
ng/mL. Growth hormone levels remained normal at less 0 0
than 0.1 ng/mL. Urinary citrate excretion also progres- Untreated Patients Control Baseline 7 mo Later
sively decreased by 19% from 463 to 373 mmol/mol cre- With GSD II
atinine (Figure 3B). Group Untreated Patients
With GSD II

ASSESSMENT OF METHYLATION CAPACITY

The integrity of methylation was assessed in a subset of
7 patients (Table 3). In 3 of these patients, plasma lev-
els of total homocysteine, SAM, and SAH, and the SAM/
SAH ratio were assayed. Homocysteine levels were nor-
mal. The levels of SAM were decreased, and the SAH levels
were elevated, resulting in a reduced ratio of SAM/SAH.
Figure 1. Urinary citrate levels from late-onset patients with glycogen storage
disease type II. A, Urine levels from 19 untreated patients with glycogen storage
disease type II compared with controls (reference upper limit, 803 mmol/mol
creatinine). B, Urine citrate at baseline and 7 months later in 4 untreated
patients illustrating the intermittent nature of excessive urine citrate levels.
All 7 patients had plasma levels of creatinine, methio-
nine, guanidinoacetate, and creatine additionally deter-

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 A P < .001 P < .001 P < .001 A B
500 250 500

Urine Citrate Level, mmol/mol Creatinine

475
225
Plasma IGF-1 Level, ng/mL
400

Plasma IGF-1 Level, ng/mL

7
450
7 200
300 425
12
175 400
200 80 Mean (SD) 154 (49) 375
43 150
41
350
100
125
325
0 100 300
30-40 41-50 51-70 Preload 100 200 300 400 Preload 100 200 300 400
Minutes After Load Minutes After Load
B P < .02 P < .001 P < .001

6000
Figure 3. Acute response to a triheptanoin meal over 6 hours by insulin-like
growth factor type 1 (IGF-1) (A) and urine citrate (B) levels. The decrease of
Plasma IGFBP-3 Level, ng/mL

5000
IGF-1 suggests increased intracellular uptake, possibly related to increased
7 adenosine triphospate availability. The rapid decline of urinary citrate is
4000 7 12
consistent with enhanced metabolism of citrate, potentially due to enhanced
3000
90 adenosine triphospate production and inactivation of adenosine
44
41 monophosphate–activated protein kinase. To convert IGF-1 to nanomoles
2000
per liter, multiply by 0.131.

1000

0 not usually affect the liver or heart. Potential hepatic ab-

30-40 41-50
Age Range, y
Figure 2. Plasma insulin-like growth factor type 1 (IGF-1) (A) and insulin-like
growth factor binding protein 3 (IGFBP-3) (B) levels from 26 untreated
patients with glycogen storage disease type II (gray bars) compared with
164 healthy participants (open bars) distributed by age ranges. The levels for
both IGF-1 and IGFBP-3 were significantly elevated for the late-onset patients
with glycogen storage disease type II at all age ranges indicating potentially
impaired intracellular transfer of IGF-1. To convert IGF-1 to nanomoles per
liter, multiply by 0.131.
mined. Plasma methionine levels were variable ranging
from 12 to 47 mM (reference range, 21-35 mM). Gua-
nidinoacetate levels were normal in all patients. How-
ever, all plasma creatinine levels were reduced below nor-
mal values, while plasma creatine levels were markedly
elevated in all 7 patients. These elevations in creatine lev-
els are too marked to be simply the consequence of in-
creased protein dietary intake.24
DISCUSSION
Other than simply muscle glycogen deposition and ve-
sicular myofiber disruption, lines of evidence for a more
extensive and complex metabolic dysfunction in GSD II
may have, in retrospect, been present previously and are
expanded in this study. Together, these findings sug-
gest an underlying energy deficiency producing a chronic
catabolic state with the potential to significantly impact
skeletal muscle function and preservation. These lines
of evidence are discussed sequentially.
RESPONSE TO DIETARY AND ERT
In GSD II, striated muscle wasting compromises physi-
cal performance and leads, ultimately, to respiratory fail-
ure. Unlike the infantile form, the late-onset disease does
51-70
normalities in GSD II have been limited to mild in-
creases in serum transaminase levels that, in fact, may
be due to myopathy. Other liver dysfunction indicators,
such as hypoglycemia and hyperammonemia, are not fea-
tures of GSD II.3 Conventional low-carbohydrate/high-
protein, calorie-balanced dietary therapy alone has not
been uniformly successful. Encouraging results have been
reported with enhanced protein/low-carbohydrate in-
take associated with a consistent physical therapy pro-
gram in the few patients studied.6,10 Enzyme replace-
ment has shown benefits in mobility and stability of the
clinical course in the first year but without reversal of
respiratory compromise or continued benefits during the
second year. Unfortunately, ERT has not been consis-
tently beneficial in all patients with GSD II.25 The lim-
ited benefits of these treatments suggest that the meta-
bolic consequences of this disorder are more extensive
than previously recognized and require further evalua-
tion to improve management in a broader metabolic con-
text.
DISPARITY IN ORGAN
INVOLVEMENT IN GSD II
Residual enzyme activity in postmortem studies is sig-
nificantly reduced in all organs tested.26 Despite the fact
that the enzyme deficiency is severe in both skeletal muscle
and liver, glycogen deposition is almost confined to skel-
etal muscle with little, if any, in liver.8 A hypothesis com-
patible with these findings is that the substrate require-
ments for the hepatic CAC and associated energy
production may be provided at the expense of skeletal
muscle protein degradation, contributing to conse-
quent deterioration. Nevertheless, these observations also
support the need to develop additional treatment strat-
egies focused on amelioration of secondary but closely
interrelated biochemical abnormalities.

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 Table 3. Plasma Indicators of Methylation Capacity in a Set of Adult-onset Patients With GSD II
Total Homocysteine,
Patient No. a nmol/L SAM, nmol/L
1 8.7 62.3
2 9.9 7.1
3 10.7 29.5
Reference range 2-14 64-125
Creatinine, mg/dL Methionine, mg/dL
1 0.4 432.7
2 0.6 358.1
3 0.2 238.7
4 0.5 462.5
5 0.2 701.3
6 0.6 328.3
7 0.3 179.1
Reference range 0.7-1.2 313.3-522.2
Abbreviations: SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.
a Normal levels are listed at the bottom of each analyte column. Patients in both sections of the table numbered equally represent the same patient. Patient
correlation with Tables 1 and 2 are (Table 3 patient number: Table 1 and 2 patient age, years): 1:40, 2:43, 3:52, 4:57, 5:58, 6:63, 7:66.
PLASMA AND URINE METABOLIC FINDINGS
Creatinine kinase was increased in 24 (73%) of the 33
patients along with occasional increases in serum trans-
aminase levels. Plasma creatinine levels were below the
reference range in all 7 patients studied. Blood acylcar-
nitine analyses revealed reduced levels of propionylcar-
nitine in 23 of 33 patients (⬍1.50 ␮M). No other evi-
dence was noted for disturbed propionate metabolism.
Reduced blood levels of propionylcarnitine can reflect
overconsumption of propionyl-coenzyme A (CoA) to aug-
ment succinyl-CoA in the CAC in an energy-deficiency
state. This decrease to less than 1.50 ␮M is often ob-
served in patients with other inherited disorders, such
as pyruvate carboxylase, adult-onset carnitine palmito-
yltransferase II, and glycogen brancher deficiencies.22,23
Urinary citrate was the only CAC intermediate that
was increased in 8 of 19 patients (Table 2). Urinary ci-
trate levels were higher than 1000 mmol/mol creatinine
in these patients (Figure 1A), exceeding normal urinary
citrate levels (typically ⬍803 mmol/mol creatinine)
(C.R.R., personal observations, August 18, 2012). De-
spite this finding, no significant difference was noted
(P = .09) from healthy controls. However, 4 of 19 pa-
tients had additional levels assayed 7 months later re-
vealing that urinary citrate levels can significantly vary
over time and are not a persistent abnormality for indi-
vidual patients. Three of these 4 patients exhibited nor-
mal citrate levels when first analyzed, which increased
significantly 7 months later, possibly reflecting changes
in their metabolic state or disease progression (Figure 1B).
When mitochondrial citrate enters the cytosol, it must
first be converted by adenosine triphosphate (ATP)–
citrate lyase (lyase) to acetyl-CoA and oxaloacetate. Acetyl-
CoA produced by the lyase reaction can be converted by
either acetyl-CoA carboxylase II (ACC II) to produce malo-
nyl-CoA (inhibiting ␤-oxidation) or be used by acetyl-
CoA carboxylase I (ACC I) to facilitate fatty acid syn-
thesis.27,28 Impairment of these reactions due to reduced
ATP availability would lead to urinary citrate excretion.
Of note, and related, adenosine monophosphate (AMP)–
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SAH, nmol/L SAM/SAH Ratio
40.9 1.53
70.8 0.10
32.6 0.90
5-20 2.1-5.6
Guanidinoacetate, mM Creatine, mg/dL
1.79 1.19
1.69 1.31
1.14 0.92
0.40 1.72
1.53 1.56
2.26 1.47
0.50 0.72
1.0-3.5 0.08-0.66
activated protein kinase (AMPK) activation by reduced
ATP stimulates cytosolic catabolic pathways that nor-
mally enhance ATP synthesis while inhibiting biosyn-
thetic reactions that consume ATP. The result is a cata-
bolic state. Active AMPK inhibits both ACC I and ACC
II, and may contribute to the intermittent excessive uri-
nary excretion of cytosolic citrate observed in patients
with GSD II.
METHYLATION DERANGEMENT IN GSD II
As observed in the adult form of GSD IV (adult polyglu-
cosan body disease),22 there is also evidence for a sec-
ondary impairment of the integrated pathways of meth-
ylation in GSD II (Table 3). These abnormalities included
a reduced SAM level, increased SAH level, elevated cre-
atine level, and reduced creatinine level. These inte-
grated pathways require ATP as depicted in Figure 4.
The increased plasma levels of creatine associated with
decreased levels of creatinine suggest compromised syn-
thesis and availability of creatine phosphate that also re-
quires ATP. This observation further supports the exis-
tence of a potential compromise of intracellular energy
metabolism. Similar abnormalities were observed in pa-
tients with GSD IV in whom, additionally (and except
for creatinine levels), normalization was observed fol-
lowing 6 months of the anaplerotic triheptanoin diet.22
ENZYME REGULATION AND GSD II
In late-onset GSD II lysosomal acid ␣-glucosidase is the
only defective enzyme involved in glycogen degrada-
tion. Although designated as a glucosidase, the enzyme
also displays acid-debrancher activity.29 Therefore, it can
catalyze the complete hydrolysis, at acid pH, of its natu-
ral substrate, glycogen. However, because the cytoplas-
mic glycogenolytic enzymes (active at neutral pH) are un-
affected in GSD II, the progressive glycogen deposition
in the cytosol as well as in lysosomes remains unex-
plained. The cytosolic glycogen accumulation might result
from continued glycogen synthesis due to ineffective regu-
WWW.JAMANEURO.COM

Methionine
THF
B12
Betaine
Methylene Folate
cycle MS
THF
Choline
5MTHF
Homocysteine
Figure 4. Schematic of the integrated methylation pathways. Blue font represents metabolites that accumulate and red font denotes those whose concentration
decreases as observed with patients with glycogen storage disease type II. ADP indicates adenosine diphosphate; AGAT, L-arginine:glycine amidinotransferase;
ATP, adenosine triphosphate; CK, creatinine kinase; GAA, guanidinoacetate; GAMT, guanidinoacetate N-methyltransferase; MS, methionine synthase; MTHF,
methyltetrahydrofolate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; and THF, tetrahydrofolate.
lation of glycogen synthase by several ATP-dependent
protein kinases, including AMPK.30-32 This could lead to
continued glycogen deposition in the cytosolic compart-
ment in excess of its degradation by the intact cytosolic
glycogenolytic enzymes. This possibility has also been
suggested for late-onset GSD IV22 and McArdle disease
(GSD type V).33
IGF-1 AND IGFBP-3 LEVELS IN GSD II
Healthy individuals receiving high-protein diets exhibit
modest increases in IGF-1 levels. For them, each 1-SD in-
crement in total protein, dairy protein, and calcium in-
take is associated with an increase in plasma IGF-1 levels
of only approximately 2.5%. However, IGFBP-3 levels are
unaffected.34,35 Plasma IGF-1 levels in patients with GSD
II were much greater at more than 84% above normal mean
levels for all age groups (P ⬍ .0001). Therefore, the sig-
nificantly elevated plasma levels of both IGF-1 and IGFBP-3
in patients with GSD II in the context of normal growth
hormone levels are not due to high-protein intake.
These extreme plasma levels of IGF-1 in GSD II may
also reflect a disturbance in nutrient sensor interactions
based on energy deficiency in muscle metabolism. The
most abundant IGF-1 binding protein is IGFBP-3, which
binds 90% of all circulating IGF-1.34 The remarkable
plasma IGF-1 elevations are compatible with dysfunc-
tional entry of IGF-1 into muscle cells via the ATP-
requiring tyrosine kinase–IGF-1 receptor. Normally, in-
tracellular IGF-1 inhibits the catabolic effects of active
AMPK via serine-threonine kinase and permits activa-
tion of the mammalian target of rapamycin. This stimu-
lates biosynthetic reactions, cell proliferation, DNA syn-
thesis, uptake of amino acids and glucose, and suppression
of proteolysis.27,36 Consistent with these observations, mice
lacking the functional IGF-1 receptor are small (45% of
normal body weight) and die soon after birth of respi-
ratory failure.37 Functional impairment of this receptor
could lead to proteolysis and progressive deterioration
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©2013 American Medical Association. All rights reserved.
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Glycine Arginine
ATP
AGAT
GAA Ornithine
SAM
GAMT
SAH Creatine
ATP
CK
Creatine ADP
phosphate
Creatinine
of muscle resulting in muscle wasting and respiratory in-
sufficiency as manifested by patients with GSD II.
Because the metabolic derangements in GSD II sug-
gest an energy-deficient catabolic state, the anaplerotic
triglyceride triheptanoin at 35% of total caloric intake has
been tested. The significant effects in a 42-year-old woman
of protein sparing, decreased proteolysis, and reversal of
acute respiratory failure coupled with a return to a nor-
mal life style have been previously described by us.38 How-
ever, IGF-1 levels were not measured. In the current study,
a 65-year-old man received a single dose of triheptanoin
(0.25 g/kg) and was evaluated over 6 hours in an at-
tempt to observe any effect of anaplerosis on plasma IGF-1
and IGFBP-3 levels and urinary citrate excretion. His
plasma IGF-1 and IGFBP-3 levels decreased to normal
levels. During the same period, his urinary citrate excre-
tion also decreased (Figure 3). These effects may reflect
the consequence of enhanced anaplerosis on the IGF-1
receptor and normalization of citrate metabolism, which
are both dependent on enhanced ATP availability. In the
context of all the results described earlier, this single ob-
servation and the related article38 provide justification for
further, systematic evaluation of anaplerotic therapy in
this disorder.
In conclusion, these observations in patients with adult-
onset GSD II suggest that there is a significant energy defi-
cit in this disease that is reflected in metabolic abnor-
malities including reduced methylation capacity. Thus,
as also suspected by others, the pathogenetic mecha-
nisms of GSD II seem to be broader than they were gen-
erally thought to be.13 The most economic interpreta-
tion of our results is that these abnormalities may be
related to compromised regulation of nutrient sensors,
producing a chronic intermittent catabolic state. These
observations, together with our prior findings involving
triheptanoin effects in GSD II and late-onset GSD IV22,38
suggest that anaplerotic diet therapy and methylation
supplements may assist in the future management of pa-
tients with GSD II.
WWW.JAMANEURO.COM
 Accepted for Publication: October 23, 2012.
Published Online: April 22, 2013. doi:10.1001/jamaneurol
.2013.1507
Correspondence: Juan M. Pascual, MD, PhD, Rare Brain
Disorders Clinic and Laboratory, The University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd,
Mail Code 8813, Dallas, TX 75390.
Author Contributions: Acquisition of data: Roe. Analy-
sis and interpretation of data: All authors. Drafting of the
manuscript: All authors. Critical revision of the manu-
script for important intellectual content: All authors. Sta-
tistical analysis: Roe. Administrative, technical, and ma-
terial support: Roe. Study supervision: Roe.
Conflict of Interest Disclosures: None reported.
Funding/Support: The study was supported in part by
the Dallas Women’s Foundation (Billingsley Fund) (Dr
Pascual), and the Baylor Health Care System Founda-
tion from generous donations of Mr William Hutchison
(Dr Roe).
Additional Contributions: Arnold Reuser, PhD, and Ans
van der Ploeg, MD, PhD, Department of Clinical Genet-
ics, Erasmus Universiteit, Rotterdam, the Netherlands,
provided untreated samples from 26 patients for this study.
Teodoro Bottiglieri, PhD, Institute of Metabolic Dis-
ease, determined SAM, SAH, methionine, homocyste-
ine, IGF-1, IGFBP-3, and GH plasma levels. Cornelis
Jakobs, PhD, Clinical Chemistry Department of the Free
University of Amsterdam, Amsterdam, the Netherlands,
analyzed the guanidinoacetate and creatine levels.
Additional Information: Part of this study was per-
formed at the Institute of Metabolic Disease, Baylor Uni-
versity Medical Center, Dallas.
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